Project Summary/Abstract The long-term objective of this application is to fully understand the biological functions of pericytes in physiological & pathological conditions and develop pericyte-based innovative therapies for various aging-related disorders, which is consistent with the mission of NIA. This proposal aims to solve a critical problem/barrier in the field of pericyte research: the lack of subpopulation-specific and age- specific/age-independent pericyte markers. We propose to screen and identify molecular markers specific for brain and muscle pericytes using a unique transgenic/biochemical approach, followed by RNAseq analyses and subsequent innovative screening/comparing strategy. In Aim 1, pericytes and vascular smooth muscle cells (vSMCs) will be isolated from the brains and skeletal muscles of Ai14:SM22?-Cre mice using FACS-based protocols optimized in our laboratory. Due to the vSMC- specific expression of tdTomato in this transgenic line, this approach, unlike others, allows separation of pericytes and vSMCs. Next, freshly isolated pericytes and vSMCs will be subjected to RNAseq analyses. The transcriptional profile of pericytes will be first negatively selected against that of vSMCs, and pericyte-enriched genes will be further compared with transcriptomes of other cells (available from public databases). Genes unique to pericytes are potential pericyte-specific markers. By comparing pericyte-specific genes from young and old mice, age-specific and age-independent markers will be identified. By comparing genes unique to brain and muscle pericytes, subpopulation-specific markers will be identified. In Aim 2, RNAseq-identified candidate genes will be validated in vitro and in vivo, and the application of validated cell-surface markers in FACS-based pericyte isolation will be assessed. Successful completion of this project will not only identify pericyte-specific markers, it will also identify subpopulation-specific (brain vs. muscle) and age-specific/age-independent (young vs. old) pericyte markers. These markers will enable isolating live pericytes at high purity for in vitro studies, targeting pericytes specifically in vivo for loss-of-function studies, and studying pericytes in a subtype-specific and age-specific/age-independent manner. These studies will dramatically enrich our knowledge in pericyte biology/function, generate new genetic tools, open doors for new lines of research, and substantially move the field forward.